Dynamic Aspect of Mechanical Stretching on the Synthesis of Cell Attachment and Spreading Factors of Human Periodontium Derived Cells.

机械拉伸对人牙周源性细胞细胞附着和扩散因子合成的动态影响。

基本信息

批准号：
11672091
负责人：
KAWASE Toshio
金额：
$ 2.3万
依托单位：
Kanagawa Dental College
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

The periodontal ligament (PL) is a typical noncalcified connective tissue that functions on tooth support and lies between cementum and alveolar bone which are mineralized tissues. PL is involved in the remodeling processes with the development of periodontium, tissue regeneration and physical stresses such as occlusion and mastication. Human periodontal ligament derived fibroblasts (HPLF), which were obtained by the explants of human periodontal tissue, were subcultured in D-MEM supplemented with 5% FCS and 0.25mM ascorbic acid-2-P.At the confluent condition. HPLF were cultured with FCS-free MCDB107 for 24 hrs. The conditioned medium of HPLF (HPLF-CM) was collected, condensed by an ultrafiltration method and fractionated with an anion exchange chromatography (IEC-DEAE column). The cell attachment and spreading factor assay was performed by using a hydrophobic well coated with proteins in HPLF-CM.SDS-PAGE revealed that one of the fraction of IEC-DEAE contained a protein with molecular weight of 40kDa as a major component. Amino acid sequence analysis using a protein sequencing system identified 40kDa protein as osteonectin (ON) (SPARC/BM-40). The expression of ON was increased by stretching. HPLF cultured in a collagen gel dramatically decreased the synthesis of ON by gel contraction. ON coated on a hydrophobic well has CASF activity. The fraction eluted with 0.28M NaCl (IEC-DEAE) contained CASFs which binded to Octyl-Sepharose column. 1%CHAPS eluted fraction was applied to SDS-PAGE and identified by the Western blotting method using monoclonal anti-ON antibody. ON was strongly interacted with hydroxyapatite which is major mineral component of cementum. The expression of ON mRNA HPLF was increased by the enamel matrix protein and HPLF-CM which were coated on a hydrophobic well and directly interacted with HPLF.ON produced by HPLF could be one of CASF and play an important role of in periodontal regeneration.

牙周韧带（PL）是一种典型的非定分结缔组织，可在牙齿支撑上起作用，并位于矿物质组织的牙髓和牙槽骨之间。 PL参与了牙周，组织再生和身体应力（例如遮挡和咀嚼）的发展过程。通过人牙周组织的外植体获得的人牙周韧带（HPLF）在融合条件下以5％FCS和0.25mm的抗坏血酸-2-p。在D-MEM中取代。用无FCS的MCDB107培养HPLF 24小时。收集HPLF（HPLF-CM）的条件培养基，通过超滤法凝结，并用阴离子交换色谱法（IEC-DEAE柱）分离。通过在HPLF-CM.SDS-PAGE中使用疏水良好涂有蛋白质的疏水性很好地进行细胞附着和扩散因子测定，表明IEC-DEAE的一部分中的一个含有40KDA的蛋白质作为主要成分。使用蛋白质测序系统的氨基酸序列分析将40KDA蛋白鉴定为骨蛋白（ON）（SPARC/BM-40）。通过拉伸增加了ON的表达。在胶原蛋白凝胶中培养的HPLF急剧减少了凝胶收缩的ON合成。在疏水井上涂层具有CASF活性。用0.28亿NaCl（IEC-DEAE）洗脱的馏分包含CASF，伴随着辛基 - 塞磷糖柱。 1％的CHAPS洗脱部分用于SDS-PAGE，并使用单克隆抗ON抗体通过蛋白质印迹方法鉴定。 ON与羟基磷灰石强烈相互作用，羟基磷灰石是牙骨质的主要矿物成分。通过在疏水性孔涂层并直接与HPLF产生的HPLF.ON相互作用的牙釉质基质蛋白和HPLF-CM增加mRNA HPLF的表达可能是CASF之一，并且在牙周再生中起重要作用。